ABSTRACT Obesity is prevalent public health problem that contributes to a rising global burden of type 2 diabetes mellitus (DM). Obesity and DM are fundamentally disorders of energy balance. Energy balance is sensed and controlled by mitochondria. Therefore, a clear understanding of mitochondrial bioenergetics in obesity may lead to new approaches to treat obesity-related health problems. Moreover, individuals with primary (genetic) mitochondrial disease develop many of the same comorbidities as those with diet-induced obesity. The shared metabolic bases for these common endocrine disturbances may represent opportunities for rational therapies. The central hypothesis of the present proposal is that abnormalities in two related mitochondrial processes, creatine metabolism and glucose utilization, contribute to the pathogenesis of insulin resistance in both primary mitochondrial disease and obesity. In both conditions, some individuals who develop insulin resistance eventually progress to DM. To test this hypothesis, we propose to perform specialized metabolomic testing on human blood samples banked in the course of Dr. McCormack's K23 mentored patient-oriented research career development award. The parent K23 project cohort includes adults ages 18 ? 65 with primary (genetic, n=15) and secondary (obesity, n=15) forms of impairment in mitochondrial oxidative phosphorylation (OXPHOS) capacity, as well as normal-weight controls (n=15). The main outcome measures in the K23 project include: magnetic resonance imaging (MRI) techniques to quantitate in vivo skeletal muscle mitochondrial OXPHOS capacity and creatine (Cr) metabolism and a stable isotope tracer- enhanced oral glucose tolerance test (OGTT*) to evaluate insulin sensitivity. For the R03, we propose 2 Aims: (1): identify a blood-based ?metabolomic signature? that is associated with altered skeletal muscle free creatine (2): to measure flux through intermediary metabolic pathways that reflect mitochondrial glucose utilization. We will test both of these measurements for their association with insulin sensitivity. In Aim 1, we focus on creatine because together with ATP derived from glucose, creatine forms phosphocreatine, an important energy reserve. We will use both targeted and untargeted metabolomics strategies to identify metabolites associated with muscle creatine as assessed by MRI. A candidate biomarker of muscle creatine will facilitate a future trial of creatine therapy. In Aim 2, we focus on mitochondrial glucose utilization because this process provides an important ?sink? for ingested glucose. We will leverage the [1-13C] oral glucose tracer used in the OGTT* to measure flux via pyruvate dehydrogenase (PDH) relative to the tricarboxylic acid (TCA) cycle. Results from this Aim will inform a future mechanistic investigation of a strategy to increase mitochondrial glucose utilization by boosting levels of a key metabolic co-factor. In summary, this R03 proposal will leverage specimens generated from the applicant's K23 parent protocol. Results will be used to support targeted clinical trials to reverse metabolic defects in individuals at risk for DM.